Pseudohypoparathyroidism (PHP) is characterized by resistance to parathyroid hormone (PTH). The most common form, PHP type Ia (PHP-Ia), is a heritable, lifelong disorder distinguished from other subtypes by having decreased activity of the alpha subunit of the guanine nucleotide-binding protein (alpha-Gs) which couples many hormone receptors to stimulation of adenylyl cyclase. Resistance to other hormones whose effects are mediated through adenylyl cyclase may occur. The Broad Objective of this proposal is to identify the molecular lesions that produce diminished function of alpha-Gs in PHP-Ia, while obtaining experience and training in molecular genetic approaches to endocrine disease. The Specific Aims of Phase I of this proposal are to: (I) Identify changes in the nucleotide sequence of the alpha-Gs gene in one pedigrees of PHP-Ia patients whose cells contain normal levels of mRNA encoding alpha-Gs; (II) Distinguish true mutations producing defective alpha-Gs from harmless nucleotide polymorphisms; (III) Determine the prevalence of the identified alpha-Gs mutation among patients with PHP-Ia and their families. The Experimental Design and Methods are: for Aim (I), to take a three- pronged approach to detecting mutations: (A) production of a cDNA library from mRNA of a PHP-Ia patient, cloning of the gene for alpha-Gs, and detection of base substitutions by RNase A protection studies or by direct sequencing of mutant cDNA; (B) amplification by the polymerase chain reaction (PCR) of the exons of the alpha-Gs gene in PHP-Ia patients, followed by sequencing; (C) amplification of the exons with annealing of GC-rich 5' tails by PCR to allow detection of single base-pair mismatches in mutant DNA: normal DNA duplexes by formamide denaturing gradient gel electrophoresis. For Aim (II), use sequence analysis, comparisons with homologous genes in family members, and transfection via retroviral vectors of putative mutant DNA into a eukaryotic expression system that genetically lacks alpha-Gs. For Aim (III), use oligonucleotides with the mutant and with normal sequence to probe gels containing size-fractionated genomic DNA from PHP-Ia patients and their family members from other pedigrees. The significance of identifying a specific genetic lesion in PHP0Ia and demonstrating its effects on alpha-Gs function is the contribution to functional mapping of alpha-Gs. Further study of alpha-Gs gene defects, based on the approaches established in this project, will promote understanding of normal hormonal signaling mechanisms as well as G-protein dysfunction in human disease.